Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia
2008; Elsevier BV; Volume: 283; Issue: 30 Linguagem: Inglês
10.1074/jbc.m801944200
ISSN1083-351X
AutoresDawei Jiang, Wenqian Chen, Jianmin Xiao, Ruiwu Wang, Huihui Kong, Peter P. Jones, Lin Zhang, Bradley R. Fruen, S.R. Wayne Chen,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoNaturally occurring mutations in the skeletal muscle Ca2+ release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca2+ release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca2+ release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process we refer to as store overload-induced Ca2+ release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca2+ activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca2+ and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca2+ activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca2+ regulation of RyR1 represents a primary causal mechanism of MH. Naturally occurring mutations in the skeletal muscle Ca2+ release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca2+ release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca2+ release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process we refer to as store overload-induced Ca2+ release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca2+ activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca2+ and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca2+ activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca2+ regulation of RyR1 represents a primary causal mechanism of MH. Malignant hyperthermia (MH) 3The abbreviations used are: MH, malignant hyperthermia; MHS, MH-susceptible; SOICR, store overload-induced Ca2+ release; wt, wild type; SR, sarcoplasmic reticulum; AM, acetoxymethyl ester; KRH, Krebs-Ringer-Hepes; FRET, fluorescence resonance energy transfer; ER, endoplasmic reticulum. 3The abbreviations used are: MH, malignant hyperthermia; MHS, MH-susceptible; SOICR, store overload-induced Ca2+ release; wt, wild type; SR, sarcoplasmic reticulum; AM, acetoxymethyl ester; KRH, Krebs-Ringer-Hepes; FRET, fluorescence resonance energy transfer; ER, endoplasmic reticulum. is an autosomal dominant, pharmacogenetic disorder of skeletal muscle. MH is triggered by volatile anesthetics (e.g. halothane) and depolarizing muscle relaxants and is characterized by muscle rigidity and a hyper-metabolic state (1Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (258) Google Scholar, 2Loke J. MacLennan D.H. Am. J. Med. 1998; 104: 470-486Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 3McCarthy T.V. Quane K.A. Lynch P.J. Hum. Mutat. 2000; 15: 410-417Crossref PubMed Scopus (299) Google Scholar, 4Lyfenko A.D. Goonasekera S.A. Dirksen R.T. Biochem. Biophys. Res. Commun. 2004; 322: 1256-1266Crossref PubMed Scopus (52) Google Scholar, 5Treves S. Anderson A.A. Ducreux S. Divet A. Bleunven C. Grasso C. Paesante S. Zorzato F. Neuromuscul. Disord. 2005; 15: 577-587Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). MH also occurs in pigs, in which it is caused by stress and known as porcine stress syndrome (6Fujii J. Otsu K. Zorzato F. de Leon S. Khanna V.K. Weiler J.E. O'Brien P.J. MacLennan D.H. Science. 1991; 253: 448-451Crossref PubMed Scopus (1238) Google Scholar, 7MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (409) Google Scholar). A single point mutation, R615C, in the pig skeletal muscle ryanodine receptor RyR1 is responsible for all cases of porcine MH (6Fujii J. Otsu K. Zorzato F. de Leon S. Khanna V.K. Weiler J.E. O'Brien P.J. MacLennan D.H. Science. 1991; 253: 448-451Crossref PubMed Scopus (1238) Google Scholar, 7MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (409) Google Scholar). On the other hand, human MH has been linked to a large number of mutations in RyR1 (4Lyfenko A.D. Goonasekera S.A. Dirksen R.T. Biochem. Biophys. Res. Commun. 2004; 322: 1256-1266Crossref PubMed Scopus (52) Google Scholar, 5Treves S. Anderson A.A. Ducreux S. Divet A. Bleunven C. Grasso C. Paesante S. Zorzato F. Neuromuscul. Disord. 2005; 15: 577-587Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 8Robinson R. Carpenter D. Shaw M. Halsall J. Hopkins P. Hum. Mutat. 2006; 27: 977-989Crossref PubMed Scopus (366) Google Scholar). Some human RyR1 mutations have also been linked to central core disease, which is often associated with MH. Although the genetic basis of MH has been well defined, the molecular mechanisms by which RyR1 mutations confer MH susceptibility and volatile anesthetics and stress trigger MH are not completely understood. The pig model has proven invaluable in investigating the molecular basis of MH, and these studies have consistently demonstrated that Ca2+ release from MH-susceptible (MHS) pig skeletal muscle or sarcoplasmic reticulum (SR) membrane vesicles is enhanced upon exposure to various stimuli (1Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (258) Google Scholar, 4Lyfenko A.D. Goonasekera S.A. Dirksen R.T. Biochem. Biophys. Res. Commun. 2004; 322: 1256-1266Crossref PubMed Scopus (52) Google Scholar, 9Otsu K. Nishida K. Kimura Y. Kuzuya T. Hori M. Kamada T. Tada M. J. Biol. Chem. 1994; 269: 9413-9415Abstract Full Text PDF PubMed Google Scholar, 10Treves S. Larini F. Menegazzi P. Steinberg T.H. Koval M. Vilsen B. Andersen J.P. Zorzato F. Biochem. J. 1994; 301: 661-665Crossref PubMed Scopus (63) Google Scholar, 11Tong J. Oyamada H. Demaurex N. Grinstein S. McCarthy T.V. MacLennan D.H. J. Biol. Chem. 1997; 272: 26332-26339Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 12Dietze B. Henke J. Eichinger H.M. Lehmann-Horn F. Melzer W. J. Physiol. (Lond.). 2000; 526: 507-514Crossref Scopus (45) Google Scholar, 13Nelson T.E. Curr. Mol. Med. 2002; 2: 347-369Crossref PubMed Scopus (79) Google Scholar, 14Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, to date no clear mechanistic basis of this enhanced responsiveness of MHS RyR1 channels to stimuli has emerged. For example, some studies reported that this enhanced activity of MHS RyR1 channels was associated with changes in the apparent sensitivity of the channel to cytosolic Ca2+ or Mg2+, whereas others found no marked difference in the apparent sensitivity to cytosolic Ca2+ activation between MHS and normal RyR1 channels (1Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (258) Google Scholar, 13Nelson T.E. Curr. Mol. Med. 2002; 2: 347-369Crossref PubMed Scopus (79) Google Scholar, 14Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 15Fill M. Coronado R. Mickelson J.R. Vilven J. Ma J.J. Jacobson B.A. Louis C.F. Biophys. J. 1990; 57: 471-475Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 16Shomer N.H. Louis C.F. Fill M. Litterer L.A. Mickelson J.R. Am. J. Physiol. 1993; 264: C125-C135Crossref PubMed Google Scholar, 17Laver D.R. Owen V.J. Junankar P.R. Taske N.L. Dulhunty A.F. Lamb G.D. Biophys. J. 1997; 73: 1913-1924Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 18Owen V.J. Taske N.L. Lamb G.D. Am. J. Physiol. 1997; 272: C203-C211Crossref PubMed Google Scholar, 19Balog E.M. Fruen B.R. Shomer N.H. Louis C.F. Biophys. J. 2001; 81: 2050-2058Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 20Murayama T. Oba T. Hara H. Wakebe K. Ikemoto N. Ogawa Y. Biochem. J. 2007; 402: 349-357Crossref PubMed Scopus (21) Google Scholar). Hence, the intrinsic properties of the RyR1 channel that are altered by the MH R615C mutation have remained undefined. Increasing evidence has highlighted the importance not only of cytosolic Ca2+, but also of luminal Ca2+, in controlling the activity of the RyR channel (21Tripathy A. Meissner G. Biophys. J. 1996; 70: 2600-2615Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 22Sitsapesan R. Williams A.J. J. Membr. Biol. 1997; 159: 179-185Crossref PubMed Scopus (114) Google Scholar, 23Gyorke S. Terentyev D. Cardiovasc. Res. 2008; 77: 245-255Crossref PubMed Scopus (179) Google Scholar). However, in comparison with the extensive investigations of the sensitivity of MHS RyR1 channels to cytosolic Ca2+ and Mg2+, the effects of the MH R615C mutation on the luminal Ca2+ sensitivity of the channel remain largely unexplored. A notable exception is found among the earliest work investigating the effect of the MH R615C mutation on Ca2+ handling by isolated SR membranes (13Nelson T.E. Curr. Mol. Med. 2002; 2: 347-369Crossref PubMed Scopus (79) Google Scholar, 24Nelson T.E. J. Clin. Investig. 1983; 72: 862-870Crossref PubMed Scopus (107) Google Scholar, 25Nelson T. Lin M. Volpe P. J. Pharmacol. Exp. Ther. 1991; 256: 645-649PubMed Google Scholar). Nelson and colleagues (13Nelson T.E. Curr. Mol. Med. 2002; 2: 347-369Crossref PubMed Scopus (79) Google Scholar, 24Nelson T.E. J. Clin. Investig. 1983; 72: 862-870Crossref PubMed Scopus (107) Google Scholar, 25Nelson T. Lin M. Volpe P. J. Pharmacol. Exp. Ther. 1991; 256: 645-649PubMed Google Scholar) originally reported that the luminal Ca2+ load required to trigger spontaneous SR Ca2+ release was markedly reduced in SR membranes isolated from MHS animals, suggesting that a defect in intraluminal Ca2+ regulation may underlie MH. We have recently shown that disease-causing mutations in the cardiac ryanodine receptor RyR2 increase the sensitivity of the channel to activation by luminal Ca2+ and enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process we have termed store overload-induced Ca2+ release (SOICR) (26Jiang D., Xiao, B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R.W. . 2004; 101: 13062-13067Google Scholar, 27Jiang D. Wang R. Xiao B. Kong H. Hunt D.J. Choi P. Zhang L. Chen S.R.W. Circ. Res. 2005; 97: 1173-1181Crossref PubMed Scopus (283) Google Scholar). Interestingly, disease-linked RyR2 mutations are located in regions corresponding to the MH/central core disease mutation regions in RyR1 (28Priori S.G. Napolitano C. J. Clin. Investig. 2005; 115: 2033-2038Crossref PubMed Scopus (76) Google Scholar). This similar distribution suggests that disease-linked RyR2 and RyR1 mutations may exert similar effects on the intrinsic properties of the channel. To test this hypothesis, in the present study, we assessed the impact of the MH R615C mutation and the MH-triggering agent, halothane, on the response of RyR1 to luminal Ca2+ and the propensity for SOICR. We found that the R615C mutation and halothane potentiated luminal Ca2+ response and SOICR. On the other hand, dantrolene, the only treatment for MH, suppressed SOICR. We propose that a reduced threshold for SOICR as a result of augmented luminal Ca2+ activation of RyR1 represents a primary defect underlying the pathogenesis of MH. Site-directed Mutagenesis—The R615C RyR1 MH mutation in the rabbit RyR1 cDNA was made by the PCR-based overlap extension method (29Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). The sequence of the PCR-amplified region was confirmed by DNA sequencing. The full-length RyR1 wt and R615C mutant cDNAs were subcloned into the mammalian expression vector pcDNA3. Single Channel Recordings—Pig RyR1 wt and R615C mutant proteins were partially purified from normal and MHS pig SR microsomes by sucrose density gradient centrifugation. Heart phosphatidylethanolamine and brain phosphatidylserine (Avanti Polar Lipids), dissolved in chloroform, were combined in a 1:1 ratio (w/w), dried under nitrogen gas, and suspended in 30 μl of n-decane at a concentration of 12 mg lipid/ml. Bilayers were formed across a 250-μm hole in a Delrin partition separating two chambers. The trans chamber (800 μl) was connected to the head stage input of an Axopatch 200A amplifier (Axon Instruments, Austin, TX). The cis chamber (1.2 ml) was held at virtual ground. A symmetrical solution containing 250 mm KCl and 25 mm Hepes (pH 7.4), was used for all recordings, unless indicated otherwise. A 4-μl aliquot (≈1 μg of protein) of the sucrose density gradient-purified wt or mutant RyR1 proteins was added to the cis chamber. Spontaneous channel activity was always tested for sensitivity to EGTA and Ca2+. The chamber to which the addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytoplasmic side of the Ca2+ release channel. The direction of single channel currents was always measured from the luminal to the cytoplasmic side of the channel, unless mentioned otherwise. Recordings were filtered at 5,000 Hz. Data analyses were carried out using the pclamp 8.1 software package (Axon Instruments). Free Ca2+ concentrations were calculated using the computer program of Fabiato and Fabiato (30Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar). Generation of Stable, Inducible HEK293 Cell Lines—Stable, inducible HEK293 cell lines expressing RyR1 wt and the R615C mutant were generated using the Flp-In T-REx core kit from Invitrogen. Briefly, the full-length cDNA encoding the RyR1 wt or mutant channel was subcloned into the inducible expression vector, pcDNA5/FRT/TO (Invitrogen). Flp-In T-REx-293 cells were then co-transfected with the inducible expression vector containing the RyR1 wt or mutant cDNA and the pOG44 vector encoding the Flp recombinase in 1:5 ratios using the Ca2+ phosphate precipitation method. Transfected cells were washed with phosphate-buffered saline (137 mm NaCl, 8 mm Na2HPO4, 1.5 mm KH2PO4, 2.7 mm KCl) 1 day after transfection and allowed to grow for 1 more day in fresh medium. The cells were then washed again with phosphate-buffered saline, harvested, and plated onto new dishes. After the cells had attached (∼ 4 h), the growth medium was replaced with a selective medium containing 200 μg/ml hygromycin (Invitrogen). The selective medium was changed every 3–4 days until the desired number of cells was grown. The hygromycin-resistant cells were pooled, aliquoted, and stored at –80 °C. These positive cells are believed to be isogenic, because the integration of the RyR1 cDNA is mediated by the Flp recombinase at a single target site. Each HEK293 cell line was tested for RyR1 expression using Western blotting analysis and immunocytofluorescence staining. Single Cell Ca2+ Imaging (Cytosolic Ca2+)—Intracellular Ca2+ transients in stable inducible HEK293 cells expressing the RyR1 wt or the R615C mutant channels were measured using single-cell Ca2+ imaging and the fluorescent Ca2+ indicator dye fura-2 acetoxymethyl ester (AM) as described previously (26Jiang D., Xiao, B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R.W. . 2004; 101: 13062-13067Google Scholar). Cells grown on glass coverslips for 24 h after induction by 1 mg/ml tetracycline (Sigma) were loaded with 5 μm fura-2 AM in Krebs-Ringer-Hepes (KRH) buffer (125 mm NaCl, 5 mm KCl, 1.2 mm KH2PO4, 6 mm glucose, 1.2 mm MgCl2, 25 mm Hepes, pH 7.4) plus 0.02% pluronic F-127 (Molecular Probes) and 0.1 mg/ml bovine serum albumin for 20 min at room temperature. The coverslips were then mounted in a perfusion chamber (Warner Instruments, Hamden, CT) on an inverted microscope (Nikon TE2000-S) equipped with an S-Fluor 20×/0.75 objective. The cells were continuously perfused with KRH buffer containing various concentrations of CaCl2 (0.2–10 mm) at room temperature. 10 mm caffeine was applied at the end of each experiment to confirm the expression of active RyR1 channels. Time lapse images (0.33 frames s–1) were captured and analyzed with the Compix Inc. Simple PCI6 software. Single Cell Ca2+ Imaging (Luminal Ca2+)—To monitor PCI6 luminal Ca2+ transients in HEK293 cells, we used the Ca2+-sensitive fluorescence resonance energy transfer (FRET)-based cameleon protein D1ER (31Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (512) Google Scholar). Stable, inducible HEK293 cells expressing RyR1 wt or mutant channels were transfected using the Ca2+ phosphate precipitation method, with D1ER cDNA 24 h before the induction of RyR1 expression. The cells were perfused continuously with KRH buffer (125 mm NaCl, 5 mm KCl, 1.2 mm KH2PO4, 6 mm glucose, 1.2 mm MgCl2, 25 mm Hepes, pH 7.4) containing various concentrations of CaCl2 (0 or 5 mm) and tetracaine (1 mm) or caffeine (20 mm) at room temperature. The images were captured with Compix Inc. Simple PCI 6 software at 470- and 535-nm emission, with excitation at 430 nm, every 2 s using an inverted microscope (Nikon TE2000-S) equipped with a S-Fluor 20×/0.75 objective. The amount of FRET was determined from the ratio of the emissions at 535 and 470 nm. The Porcine MH R615C Mutation Enhances the Luminal Ca2+ Activation of Single RyR1 Channels—To directly assess the impact of the R615C mutation on luminal Ca2+ activation, we incorporated single normal (RyR1 wt) and MHS (RyR1-R615C) RyR1 channels into planar lipid bilayers and examined their responses to increasing concentrations of luminal Ca2+. As shown in Fig. 1A, elevating the luminal Ca2+ concentration from ∼45 nm to 50 mm had little effect on single wt channels but markedly activated single R615C mutant channels with an activation threshold of about 0.5 mm luminal Ca2+ (Fig. 1A, panel e). For instance, at 1.2 mm luminal Ca2+, the average open probability (Po) for single R615C mutant channels was 0.070 ± 0.024 (n = 14) (Fig. 1A, panel d), which was significantly greater than that of single wt channels (0.001 ± 0.0004, n = 8) (p < 0.05) (Fig. 1A, panel b). These observations directly demonstrate that the R615C mutation enhances the response of the RyR1 channel to luminal Ca2+ activation. The R615C Mutation Has Little Effect on the Cytosolic Ca2+ Dependence of Single RyR1 Channels—We next determined the response of wt and R615C to cytosolic Ca2+ using single channel recordings. As shown in Fig. 1B, both single wt and R615C channels were maximally activated by ∼10 μm cytosolic Ca2+ with an activation threshold about ∼100 nm and were completely inhibited by ∼5 mm Ca2+. Although the extent of maximum activation of the R615C channels by cytosolic Ca2+ was greater than that of the wt channels (Fig. 1B, panel a), the cytosolic Ca2+ dependence of activation or inactivation of the wt and R615C channels was similar, as seen from the normalized Ca2+ responses (Fig. 1B, panel b). These observations indicate that the R615C mutation does not markedly alter the sensitivity of single RyR1 channels to cytosolic Ca2+ activation or inactivation. Halothane Potentiates the Luminal Ca2+ Response of Single RyR1 Channels—The effect of halothane on the luminal Ca2+ activation of single RyR1 channels was also investigated (Fig. 2). A single R615C mutant channel exhibited little activity in the presence of 45 nm cytosolic and luminal Ca2+ (Fig. 2A). The addition of 600 μm luminal Ca2+ slightly activated the channel (Fig. 2B). A subsequent addition of 20 mm halothane to the cytosolic side of the channel markedly increased the channel activity (Fig. 2C). The average Po after the addition of halothane was 0.176 ± 0.049 (n = 6) in the presence of 600 μm luminal Ca2+, which was significantly greater than that before the addition of halothane (0.030 ± 0.004) (n = 6) (p < 0.03). It should be noted that halothane has been shown to have little effect on the apparent affinity of EGTA for Ca2+ (32Housmans P.R. Wanek L.A. Anal. Biochem. 2000; 284: 60-64Crossref PubMed Scopus (3) Google Scholar). Importantly, this halothane-induced enhancement was dependent on the presence of luminal Ca2+. Reducing the luminal Ca2+ from 600 μm to ∼35 nm decreased the channel activity to the basal level (Fig. 2D). Similarly, halothane also activated single RyR1 wt channels in a luminal Ca2+-dependent manner (not shown). It should be noted that because of its highly volatile nature, the actual concentration of halothane in the bilayer recording solution would be much lower than that added to the chamber. Nevertheless, these data indicate that at low cytosolic Ca2+, halothane potentiates the response of RyR1 to luminal Ca2+. Halothane and the R615C Mutation Enhance the Propensity for SOICR in HEK293 Cells—It has long been demonstrated that halothane induces spontaneous contracture in MHS muscle, but not in normal muscle in an external Ca2+-dependent manner (33Nelson T.E. Bedell D.M. Jones E.W. Anesthesiology. 1975; 42: 301-306Crossref PubMed Scopus (39) Google Scholar), and that the halothane-induced spontaneous SR Ca2+ release is dependent on the SR Ca2+ load (34Ohnishi S.T. Taylor S. Gronert G.A. FEBS Lett. 1983; 161: 103-117Crossref PubMed Scopus (118) Google Scholar). To determine whether halothane and the R615C mutation also augment RyR1-mediated spontaneous Ca2+ release or SOICR in a nonmuscle environment, we generated stable, inducible HEK293 cell lines expressing wt or R615C. We have previously shown that elevated [Ca2+]o induces SOICR in HEK293 cells expressing RyR2 (26Jiang D., Xiao, B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R.W. . 2004; 101: 13062-13067Google Scholar, 27Jiang D. Wang R. Xiao B. Kong H. Hunt D.J. Choi P. Zhang L. Chen S.R.W. Circ. Res. 2005; 97: 1173-1181Crossref PubMed Scopus (283) Google Scholar). Unlike RyR2-expressing cells, cells expressing RyR1 wt or the R615C mutant did not show SOICR in response to elevated [Ca2+]o in the absence of stimuli (Fig. 3A, panel b). However, in the presence of low concentrations of halothane, elevated [Ca2+]o triggered SOICR in both RyR1 wt- and R615C mutant-expressing HEK293 cells (Fig. 3A, panels a and b). Analyzing a number of oscillating cells revealed that HEK293 cells expressing R615C displayed a greater propensity for SOICR than cells expressing wt (Fig. 3A, panel b). The frequency of Ca2+ oscillations in cells expressing the R615C mutant was also much higher than that in cells expressing wt (Fig. 3A, panel c). Similar results were obtained when halothane was replaced with caffeine (Fig. 3A, panels d and e). The parental HEK293 cells do not express a detectable level of RyRs (26Jiang D., Xiao, B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R.W. . 2004; 101: 13062-13067Google Scholar, 35Tong J. Du G.G. Chen S.R. MacLennan D.H. Biochem. J. 1999; 343: 39-44Crossref PubMed Google Scholar). These observations indicate that the MH R615C mutation, halothane, and caffeine can enhance the propensity for SOICR in a nonmuscle environment, suggesting that the response of RyR1 to Ca2+ overload is a major determinant of halothane- or caffeine-induced spontaneous Ca2+ release. The R615C Mutation Reduces the Luminal Ca2+ Threshold at Which SOICR Occurs—To directly measure the luminal Ca2+ threshold at which SOICR occurs, we used a FRET-based endoplasmic reticulum (ER) Ca2+ sensor protein, D1ER (31Palmer A.E. Jin C. Reed J.C. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17404-17409Crossref PubMed Scopus (512) Google Scholar), to monitor the ER luminal Ca2+ dynamics during store Ca2+ overload in HEK293 cells expressing RyR1 wt or R615C. As shown in Fig. 3B (panels a and b), elevated [Ca2+]o increased the level of ER luminal Ca2+. When the luminal Ca2+ reached a threshold level, SOICR occurred, displaying as downward deflections in the FRET signal. SOICR was then suppressed by 1.0 mm tetracaine, an inhibitor of RyR1, to estimate the maximum luminal Ca2+ level. Caffeine (20 mm), an activator of RyR1, was then used to estimate the minimum luminal Ca2+ level by emptying the store. Fig. 3B (panel c) shows that the luminal Ca2+ threshold (percentage of maximum luminal Ca2+ store capacity) at which SOICR occurs is significantly lower in cells expressing R615C (76.1 ± 0.8%, n = 243) than in cells expressing wt (89.5 ± 1.0%, n = 106) (p < 0.00001) in the presence of 2 mm caffeine. There was no significant difference in the maximum luminal Ca2+ store capacity between the R615C-expressing cells (105.5 ± 6.2%) and the wt-expressing cells (100%) (p = 0.25), which was calculated by subtracting the minimum FRET signal (in the presence of 20 mm caffeine) from the maximum FRET signal (in the presence of 1 mm tetracaine). Similarly, the luminal Ca2+ threshold at which SOICR occurs is significantly lower in cells expressing R615C (80.0 ± 3.1%, n = 107) than in cells expressing wt (93.2 ± 0.8%, n = 108) (p < 0.00001) in the presence of 2 mm halothane. The resting luminal Ca2+ level in the presence of near 0 mm external Ca2+ and 2 mm caffeine is also lower in cells expressing R615C (34.0 ± 2.9%) than in cells expressing wt (54.5 ± 4.5%) (p < 0.00001). Similarly, the resting luminal Ca2+ level in the presence of near 0 mm external Ca2+ and 2 mm halothane is lower in cells expressing R615C (62.8 ± 3.3%) than in cells expressing wt (81.5 ± 2.4%) (p < 0.00001). Taken together, these data are consistent with those of single channel studies showing that the R615C mutation and halothane enhance the response of RyR1 to luminal Ca2+, leading to a reduced SOICR threshold and resting ER Ca2+ level. Dantrolene Abolishes SOICR in HEK293 Cells Expressing RyR1-R615C but Not in Cells Expressing RyR2-N4104K—It has also long been shown that dantrolene suppresses caffeine- or halothane-induced spontaneous contracture in MHS muscle (36Okumura F. Crocker B.D. Denborough M.A. Br. J. Anaesth. 1980; 52: 377-383Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 37Foster P.S. Denborough M.A. Br. J. Anaesth. 1989; 62: 566-572Abstract Full Text PDF PubMed Scopus (10) Google Scholar) and that dantrolene inhibits spontaneous Ca2+ release from skeletal muscle SR but not from cardiac muscle SR (38Van Winkle W.B. Science. 1976; 193: 1130-1131Crossref PubMed Scopus (203) Google Scholar). To determine whether dantrolene can also suppress caffeine-induced spontaneous Ca2+ release in a nonmuscle environment, we assessed the impact of dantrolene on SOICR in HEK293 cells. As shown in Fig. 4, HEK293 cells expressing the RyR1-R615C mutant exhibited Ca2+ oscillations in the presence of 5 mm [Ca2+]o plus 2.0 mm caffeine. The addition of 100 nm dantrolene diminished these oscillations, reducing the number of oscillating cells by ∼80% (Fig. 4, A and B). Fig. 4C shows that dantrolene suppressed SOICR in HEK293 cells expressing either the R615C mutant or RyR1 wt with an IC50 of ∼10–20 nm. Interestingly and in contrast, dantrolene did not abolish Ca2+ oscillations in HEK293 cells expressing the disease-causing RyR2 mutation, N4104K, even at high concentrations (10 μm) (Fig. 4D). These data demonstrate that dantrolene potently suppresses RyR1-mediated, but not RyR2-mediated, SOICR in HEK293 cells and therefore suggest that the inhibition of RyR1-mediated SOICR may represent a primary therapeutic action of dantrolene. Investigations over the past decades have greatly advanced our understanding of the molecular and cellular mechanisms of MH. However, some fundamental questions still remain, including: 1) what intrinsic properties of the RyR1 channel altered by mutations are principally responsible for MH pathogenesis? 2) how do volatile anesthetics trigger MH? and 3) how does dantrolene suppress MH? In the present study, we demonstrated that both the MH R615C mutation and MH-triggering agent, halothane, sensitize the RyR1 channel to activation by luminal Ca2+ and reduce the threshold for SOICR. In contrast, dantrolene, an effective treatment for MH, suppresses SOICR. Based on these observations, we propose that volatile anesthetics, by further reducing the already reduced threshold for SOICR in the MHS muscle, trigger spontaneous Ca2+ release and thus MH, whereas dantrolene suppresses spontaneous Ca2+ release and MH by increasing the SOICR threshold. The MH R615C Mutation Sensitizes the RyR1 Channel to Luminal Ca2+ Activation—Despite the consistent observation that the MHS pig RyR1 channel is more active than the normal RyR1 channel upon stimulatio
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